Published on Web 02/23/2008

Inhibition of Helicobacter pylori Aminoacyl-tRNA Amidotransferase by

Puromycin Analogues
Christian Balg,† Jonathan L. Huot,‡ Jacques Lapointe,‡ and Robert Chênevert*,†
Département de Chimie and Département de Biochimie et de Microbiologie, Centre de Recherche sur la Fonction,
la Structure et l’Ingénierie des Protéines (CREFSIP), Faculté des Sciences et de Génie,
UniVersité LaVal, Québec, Canada, G1K 7P4
Received November 6, 2007; E-mail: robert.chenevert@chm.ulaval.ca

The biosynthesis of aminoacylated tRNAs (aa-tRNA) is a critical

step in the faithful translation of the genetic code into proteins. In
most organisms, the 20 amino acids (aa) are esterified to their
corresponding tRNA by 20 different aminoacyl-tRNA synthetases Figure 1. Indirect pathway for Gln-tRNAGln and Asn-tRNAAsn biosynthesis.
(aaRS), each of which is specific for one amino acid and a
corresponding set of tRNAs (direct aminoacylation pathway).1
Recent genomic studies revealed the absence of glutaminyl-tRNA
synthetase (GlnRS) and/or asparaginyl-tRNA synthetase (AsnRS)
in archaebacteria, Gram-positive eubacteria, and many Gram-
negative eubacteria. The survival of microorganisms missing one
or both of these essential enzymes implies an alternative pathway
for the formation of Gln-tRNAGln and Asn-tRNAAsn. This indirect
pathway involves the misacylation of tRNAGln with Glu (or tRNAAsn
with Asp) by a nondiscriminating aminoacyl-tRNA synthetase (ND-
aaRS) and the subsequent transamidation of the misacylated aa-
tRNA by an amidotransferase (AdT) (Figure 1).2
The dissemination of antibiotic resistance has become a major
problem in clinical medicine, and there is a critical need to develop
antibacterial agents with novel modes of action.3 The widespread Figure 2. Reaction mechanism: Glu-tRNAGln amidotransferase (GluAdT),
n ) 2, tRNAaa ) tRNAGln. Asp-tRNAAsn amidotransferase (AspAdT), n )
use of the indirect transamidation pathway among prominent human 1, tRNAaa ) tRNAAsn.
pathogens4 and its absence in the mammalian cytoplasm identify
AdT as an interesting target for the development of new and highly recently, only a few AdT inhibitors have been reported in part
specific antimicrobial agents.5 Here we report the synthesis and because of the absence of a convenient assay. Some analogues of
biological evaluation of puromycin analogues as mechanism-based glutamine and ATP were useful to study the reaction mechanism,7
inhibitors of aminoacyl-tRNA amidotransferases. but these inhibitors are likely to interfere with many other enzymes
The proposed mechanism for the transamidation reaction cata-
acting on the same substrates.
lyzed by amidotransferases is a three-step event (Figure 2). First,
The general synthetic approach to the puromycin analogues is
the hydrolysis of the amido donor, glutamine, forms glutamic acid
outlined in Scheme 1 (see Supporting Information for full details
and enzyme-bound NH3 (glutaminase step). The second step is the
of each synthesis). The coupling of puromycin aminonucleoside 1
activation of the side-chain carboxyl group of the amino acid fixed
with N-tert-butyloxycarbonyl (N-Boc) protected amino acids 2 using
on the tRNA (Glu-tRNAGln or Asp-tRNAAsn) resulting from the
N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydrox-
reaction of this carboxyl group with ATP to form a mixed anhydride
ysuccinimide (NHS) provided amides 3 in high yields (72%-91%)
(kinase step). In this intermediate, the high-energy anhydride bond
despite the presence of two unprotected hydroxyl groups on the
activates the carboxyl group. Finally, the aminolysis of the activated
ribose. Removal of the Boc protecting group under standard
amido acceptor by enzyme-bound NH3 (transamidase step) forms
conditions with CF3COOH gave products 4, but glycosyl bond
the final product (Gln-tRNAGln or Asn-tRNAAsn). The overall
cleavage and depurination lowered the yields. In contrast, Boc
reaction is the simple conversion of the side chain carboxylic acid
removal with 4 M HCl/dioxane proceeded without difficulty to
(Glu or Asp) into an amide (Gln or Asn) while the amino acid is
furnish the final product 4 in high yields (89%-100%).
still attached to a tRNA (pretranslational modification).
Two types of amidotransferases have been identified so far in With a series of compounds in hand, we set out to evaluate their
nature. GatCAB are heterotrimeric proteins encoded by genes inhibitory activities against Helicobacter pylori GatCAB amidot-
named gatC, gatA, and gatB. These enzymes found in both archaea ransferase (Table 1). Enzyme production and kinetic experiments
and bacteria can transamidate both Glu-tRNAGln and Asp-tRNAAsn. were carried out as previously described.8 Competitive inhibition
The second type, heterodimeric GatDE, occurs only in archaea and of H. pylori AdT by compounds 4a-4h, with respect to Asp-
functions solely as a GluAdT. The first crystal structures of tRNAAsn, was characterized in the presence of saturating concentra-
members of each type have been determined recently.6 Up to tions of the two other substrates (2 mM ATP and 1.28 mM
L-glutamine) and of 0.50-1.25 µM Asp-tRNAAsn. The decrease of
† Department of Chemistry.
the transamidation rate from its value Vo in the absence of inhibitor,
‡ Department of Biochemistry and Microbiology. to its value Vi in the presence of various inhibitor concentrations,
3264 9 J. AM. CHEM. SOC. 2008, 130, 3264-3265 10.1021/ja7100714 CCC: $40.75 © 2008 American Chemical Society

Scheme 1. General Synthetic Approach to Inhibitorsa
a Conditions: (a) EDC, N-hydroxysuccinimide, DMF, 72-91%; (b) 4
M HCl, dioxane, 89-100%.
Table 1. Inhibition of Helicobacter pylori GatCAB
Amidotransferase
was used to identify the competitive nature of the inhibitor and to
obtain the Ki value, as described in the Supporting Information (see
Figure S1).
We initially assayed the parent compound puromycin 4a, an
aminonucleoside antibiotic produced by Streptomyces alboniger.
This natural product mimics the charged 3′-terminus of aminoa-
cylated tRNA and has been widely used as a basic tool for the
study of protein synthesis on the ribosomes. Puromycin is a very
weak inhibitor of AdT (Ki ) 4 mM).
The amino acid chain of puromycin is related to tyrosine and
differs from the aspartic and glutamic side chains transformed by
AdT (Figure 2). Replacement of the methoxyphenyl moiety of
puromycin by carboxylic acid derivatives considerably enhances
the ability to inhibit AdT. Compounds 4b and 4c, analogues of the
3′-ends of Asp-tRNA and Glu-tRNA, are competitive inhibitors
with similar Ki values of 134 and 105 µM, respectively. This is
consistent with the fact that the enzyme is equally efficient in
transamidation of both Asp-tRNAAsn and Glu-tRNAGln.9
Replacement of the R-amino group by a hydroxyl (4d) slightly
increases the Ki (Ki ) 105 µM for 4c vs 130 µM for 4d). The
amide variant 4e has structural homology to the final product of
the enzymatic reaction and shows improved activity (Ki ) 45 µM)
over the carboxylic acid analogues 4b and 4c.
COMMUNICATIONS
Various phosphorus- and sulfur-containing derivatives have been
previously proposed as analogues of tetrahedral intermediates
formed transiently during enzymatic reactions involving formation
or hydrolysis of amide bonds.10 As stable analogues of the transition
state in the last step of the transamidation process, we designed 4f,
4g, and 4h where the carbonyl to be attacked by ammonia is
replaced by a tetrahedral phosphorus or sulfur atom with a methyl
group mimicking ammonia. Racemic 4f prepared from (D,L)-
phosphinothricin did inhibit transamidase activity with a Ki ) 33
µM. Likewise, a Ki ) 11 µM was determined for the diastereo-
merically mixed L-methionine (R,S)-sulfoxyde 4g. The sulfone 4h
exhibited the highest activity with a Ki ) 4 µM. Competitive
inhibition with respect to Asp-tRNAAsn was observed for all
puromycin analogues.
In conclusion, we have identified analogues of the natural product
puromycin that have inhibitory activity against bacterial aminoacyl-
tRNA amidotransferases. These mechanism-based inhibitors will
provide useful chemical probes for further mechanistic investiga-
tions, ligands for X-ray crystallography, and a potential avenue to
develop antibiotics with a novel mode of action.11 Investigations
along these lines are currently underway.
Acknowledgment. This work was supported by the Natural
Sciences and Engineering Research Council of Canada (NSERC)
and the “Fonds de recherche sur la nature et les technologies,
Québec (FQRNT)”.
Supporting Information Available: Experimental procedures and
characterization data for all intermediates and final products; figures
of 1H and 13C NMR spectra. This material is available free of charge
via the Internet at http://pubs.acs.org.
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JA7100714
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